1. Field of the Invention
The present invention relates generally to nanostructured matrices, and more particularly to the fabrication and use of nanostructured matrices for separation and analysis of molecules.
2. Description of the Prior Art
Polyacrylamide gel electrophoresis (PAGE) remains the standard for protein separation and identification in biotechnology. Nevertheless, the set of separation strategies that rely on this technique are hampered by: (1) inconvenience of preparation of the variety of gels needed for the separations, (2) inherent inconsistencies in production conditions; and therefore, irreproducibility between different batches of gels, (3) limited resolution and dynamic range of biomolecular separations, (4) susceptibility of the polymer to degradation under high electric fields, (5) lack of reusability, and (6) difficulty in incorporation of these techniques into strategies for development of multi-dimensional (multi-technique) integrated separation systems.
Gradient PAGE techniques are recognized to have the potential to have excellent resolution and dynamic range, but their utility is greatly hampered by the need for cumbersome gel preparation protocols and lack of reproducibility.
The demand for precise separation of molecules using small sample volumes is increasing. Separation of molecules across matrices or membranes has been known for long in the art. Separations are generally achieved by employing barriers that allow cut-offs at a precise molecular weight or by size-exclusion. The art describes structures where molecular transport and filtration take place perpendicular to the surface of the separating material. The currently available systems, however, suffer from a number of drawbacks. For example, biomolecules may not be amenable to separation by many of the available systems. For example, reaction steps may denature or inactivate the molecules themselves. The matrices formed are generally composed of non-uniform structures. Even where a gradation in size of structures is required, they may be random or at best have to be serially and sequentially arrayed through a cumbersome process of lithography. Fabrication of such separation devices also pose problems in terms of batch-to-batch variations and consequently poor reproducibility of results therefrom. Lack of efficiency of separation or loss of sample volume are also encountered.
Nano-filtration of molecules using xe2x80x9cBrownian ratchetsxe2x80x9d in which assymetric diffusion leads to separation of molecules based on their size (van Oudenaarden et al. Science, 285: 1046-1052, 1999) has been tried with some success. Chou et al., Proc. Natl. Acad. Sci. 96, 13762-13765, 1999, attempted separation of DNA molecules using microsystems formed by conventional photolithography. However, the developments have not gained ground with users primarily because of the difficulty of preparation of the nanofluidic systems and the associated high-cost of fabrication. Other separation matrices such as gradient polyacrylamide gels, where one-dimension filtration was achieved by manipulating pore-size through control of cross-linker, monomer and solvent concentrations, has shown limited success. Even though the separation is effective, the preparation process is tedious and the results obtained are not reproducible. xe2x80x9cArtificial gelsxe2x80x9d incorporating regular arrays of nanoscale pillars created through electron beam and/or imprint lithography have been described, for example, in U.S. Pat. No. 6,110,339 to Brueck et al. and by Turner et al. (J. Vac. Sci. Technol. B., 16 3835-3840, 1998). All these nanolithographically-defined structures utilize regular arrays of uniform-sized nanostructures throughout the separation matrix. Thus, the systems suffer from resolution and flexibility limitations. It is also difficult to integrate such a system with other more complex separation devices. Thus, the need for an efficient, highly-resolving, flexible, cost-efficient and reproducible molecular-separation matrix, is largely unmet.
It is therefore an object of the present invention to provide a highly-efficient and facile nanostructured matrix for separation and analysis of molecules.
It is a further object of the present invention to provide a matrix that enables gradient or non-uniform transport of molecules across a plane parallel to the surface of the matrix.
A further object of the present invention is to enable integration of multi-dimensional multi-technique molecular separation systems into a single platform.
Yet another object of the present invention is to provide for customized fabrication of a nanostructured separation matrix including an array having a gradient property.
It is yet another object of the present invention is to provide a nanostructured matrix that may be easily cast to cater to different ranges of molecular separations, in terms of resolution and dynamics.
Another object of the present invention is to enable uniform consistency in the composition of the nanostructures forming the separation matrix.
Yet another object of the present invention is to enable separation and/or identification of a molecular species.
A further object of the present invention is to enable calibration-free use of the separation/analysis process.
Yet another object of the present invention is to enable multiple use of a single separation matrix.
A further object of the present invention is to enable parallel production of separation matrices at relatively low cost.
In all of the above embodiments, it is an object to provide enhanced reproducibility and resolution in the separation of molecules.
According to a first broad aspect of the present invention, there is provided a matrix comprising an array of nanostructures arranged so that the array has a gradient property.
According to second broad aspect of the invention, there is provided a method for forming an array having a gradient property comprising the steps of: (a) providing a substrate; and (b) forming nanostructures on the substrate to form an array having a gradient property.
According to a third broad aspect of the invention, there is provided a separation method comprising the steps of: (a) providing a matrix comprising an array having a gradient property, the array comprising nanostructures; and (b) conducting at least one biomolecule separation process to separate biomolecules in a composition containing a plurality of biomolecules using the matrix.